Proteases, which catalyze the hydrolysis of amide bonds in peptides and proteins, play essential roles in most biological processes and are very important therapeutic targets for a multitude of diseases, including cancer, viral, parasitic, and bacterial infections, inflammation and cardiovascular disease. Indeed, well over a thousand proteases are predicted to be encoded by the human genome and those of other organisms. 29-31 To derive the full value of this gene sequence information the function of these newly identified proteases must be determined. In this grant, we propose to develop efficient and systematic combinatorial tools to determine the substrate specificity profiles of any protease. Knowledge of substrate specificity greatly facilitates the identification of natural substrates of proteases and can provide key information for the design of potent and selective protease inhibitors. In addition, such specific substrates can be employed to dissect biological pathways and visualize protease activity in cells. SPECIFIC AIM I: To develop fluorogenic substrate microarray approaches to rapidly obtain substrate specificity profiles for serine and cysteine proteases. SPECIFIC AIM II: To develop thioester-based positional scanning library and microarray approaches to rapidly obtain substrate specificity profiles for aspartyl proteases and metalloproteases. SPECIFIC AIM III: To develop fluorogenic substrates to monitor protease activity in cells. Specifically, we will use fluorogenic substrates to study neutrophil-mediated cell killing. During the previous grant cycle we developed several inhibitor libraries as well as potent inhibitors to specific proteases. A number of leading biological researchers are currently using these libraries and inhibitors. We will also continue to support these collaborative efforts.